Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, at least three different TGase enzymes, TGases 1, 2 and 3, are expressed. They cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. The partitioning between the cytosol and membranes is controlled by differential acylation by myristate and palmitate of a cluster of cysteine residues located on a membrane anchorage amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have tested several available methods for the large-scale expression of this enzyme, which to date have not been successful, although we are able to produce microgram amounts of enzyme which can be stored indefinitely. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We have expressed in baculovirus several of the known mutations to explore the bases of loss of enzyme activity. These studies have been coupled with structural analyses based on comparative structure with the TGase 3 and factor XIIIa enzymes. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. For example, some mutations clearly suggest that lamellar ichthyosis disease may be caused by either insufficient enzyme activity, or an enzyme form that is not appropriately postsynthetically modified and cannot be utilized by the cell. In addition, these studies offer the only opportunity to date to understand its structure and substrate specificity. Transglutaminase 2 Our main focus of this enzyme is to obtain atomic resolution structural information by X-ray diffraction of crystals. To date, we have developed methods for the large scale preparation of the active enzyme in baculovirus. Ongoing work will attempt to purify active forms of the enzyme in order to initiate crystallization trials. Transglutaminase 3 The TGase 3 enzyme is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. In addition, data from this laboratory have shown that it is the preferred enzyme for cross-linking in vivo of several important substrates involved in barrier or other functions, including loricrin, small proline rich proteins, and trichohyalin. We have developed methods for the large-scale expression and purification of several forms of the TGase 3 enzyme in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Each of these has been crystallized and the structures have been resolved by X-ray diffraction methods to 2.5 A or less. Each possess 2 or 3 calcium ions that are required for activity. Several residues have identified at or near the active site region that may confer substrate specificity for the enzyme. These studies will continue. Hopefully, they will allow us to rationally design specific inhibitors for this enzyme. Expression of TGases in non-epithelial tissues: roles in degenerative diseases By RT-PCR methods, we have found that TGases 1, 2 and 3 are widely expressed in a variety of non-epithelial tissues, including in particular, various tissues within the brain, connective tissues, fibroblasts and muscle. Heretofore, these tissues were thought to express only the cytosolic TGase 2 enzyme. The expression of these enzymes has been confirmed by both immunoprecipitation and indirect immunofluorescence methods using specific probes. Moreover, mRNA and enzyme levels of TGases 1 and 3 are up-regulated in pathological conditions, including Alzheimer?s Disease (AD) and sporadic inclusion body myositis (SIBM). We found co-localization of the TGase enzyme antigens with the inclusion bodies/senile plaques of both diseases. In addition, we isolated insoluble proteins from tissue slices from these AD and SIBM which contained substantial amounts of isopeptide cross-link formed by TGases, suggesting a direct causative link. Also, in the case of SIBM, we performed sequencing analyses to demonstrate the presence of significant amounts of beta-amyloid protein cross-linked to itself, myosin and desmin muscle proteins. Together, these data demonstrate that elevated levels of TGases 1 and/or 3 correlate with disease pathogenesis and contribute directly to the formation of insoluble cross-linked bodies that interfere with normal cellular function and thus degenerative disease. Depending on availability of adequate amounts of tissue, further work will be directed toward analysis of the forms of the TGase 1 and 3 enzymes and more detailed sequencing analyses. We are performing parallel studies with other diseases including celiac disease, type II diabetes and Huntington?s Disease, all of which have aberrantly high levels of TGase expression. In addition, three new members of the TGase family have been discovered recently. The expression of these will also be tested in these disease states. We anticipate that such studies may provide valuable new insights into both disease etiology as well as methods to control the degenerative disease processes.